Structural systems biology - the combination of system biology with structural biology, has emerged as a powerful tool for understanding complex systems significant for human health. DNA replication and repair are essential life processes critical for genome integrity. While structures of many individual replication proteins have been solved, larger replisomal assemblies still present challenges for conventional structural methods. Thus, an excellent opportunity exists to apply structural systems biology approaches to characterize dynamic replication complexes. The proposal is focused on the assemblies of the core replication protein DNA ligase I (Lig1) with partners Flap Endonuclease 1 (FEN1), Proliferating Cell Nuclear Antigen (PCNA) and Rad9-Hus1- Rad1 (9-1-1) on DNA. We will delineate the mechanisms for coordinated enzyme exchange on PCNA (9-1-1), important for genome duplication and maintenance. To define inherently dynamic complexes, we combine advanced computational methods with structural techniques that can collect data on flexible macromolecular systems - small angle X-ray scattering (SAXS), electron microscopy (EM) and single-molecule Frster Resonance Energy Transfer (smFRET). Known high-resolution structures of constituents in these complexes will be integrated with SAXS, EM, smFRET and biochemical data to yield information on the larger assemblies through hybrid computational protocols we are developing. Such close interplay of computation and experiment is needed for analysis of dynamic assemblies. Our experimental work is stronger with molecular- level models to interpret the data; and our computational work will benefit from diverse experimental techniques to restrain and cross-validate the models.
In Aim1 we will delineate how Lig1 recognizes substrate DNA.
In Aim2 we will unravel the origins of Lig1/DNA stabilization by PCNA/9-1-1 and model ternary Lig1/clamp/DNA complexes.
In Aim3 we will synthesize diverse structural information to discover the controlling elements for coordinated handoff of DNA between FEN1 and Lig1. Our strategy is to address these key mechanistic questions relevant to cancer etiology and interventions, through synergistic biochemical, structural and hybrid modeling methods. The results will clarify mechanisms whereby loss of function of these coordinated replication complexes may lead to inheritable genetic diseases or cancer susceptibility.
DNA replication is a major target for cancer therapies, while efficient repair antagonizes those same therapies. Both replication and repair are critically dependent on the dynamics, coordinated access, and conformational switching of key proteins in these processes. We will model and structurally characterize dynamic assemblies of these proteins to elucidate their roles in coordinating replication and repair activities. Success of thi research will have impact on fundamental understanding of cancer etiology.
Showing the most recent 10 out of 16 publications
